R-PTP-κ Inhibits Contact-Dependent Cell Growth by Suppressing E2F Activity.

Density-dependent regulation of cell growth is presumed to be caused by cell-cell contact, but the underlying molecular mechanism is not yet clearly defined. Here, we report that receptor-type protein tyrosine phosphatase-kappa (R-PTP-κ) is an important regulator of cell contact-dependent growth inhibition. R-PTP-κ expression increased in proportion to cell density. siRNA-mediated R-PTP-κ downregulation led to the loss of cell contact-mediated growth inhibition, whereas its upregulation reduced anchorage-independent cell growth in soft agar as well as tumor growth in nude mice. Expression profiling and luciferase reporter system-mediated signaling pathway analysis revealed that R-PTP-κ induced under cell contact conditions distinctly suppressed E2F activity. Among the structural domains of R-PTP-κ, the cytoplasmic domain containing the tandemly repeated PTP motif acts as a potent downregulator of the E2F pathway. Specifically, R-PTP-κ suppressed CDK2 activity through the induction of p21Cip1/WAF-1 and p27Kip1, resulting in cell cycle arrest at the G1 phase. In transcriptome-based public datasets generated from four different tumor types, R-PTP-κ expression was negatively correlated with the expression pattern and prognostic value of two known E2F1 target genes (CCNE1 and CDC25A). Therefore, our results indicate that the R-PTP-κ-E2F axis plays a crucial role in cell growth-inhibitory signaling arising from cell-cell contact conditions.

Synaptotagmin 11 scaffolds MKK7-JNK signaling process to promote stem-like molecular subtype gastric cancer oncogenesis.

BACKGROUND: Identifying biomarkers related to the diagnosis and treatment of gastric cancer (GC) has not made significant progress due to the heterogeneity of tumors. Genes involved in histological classification and genetic correlation studies are essential to develop an appropriate treatment for GC. METHODS: In vitro and in vivo lentiviral shRNA library screening was performed. The expression of Synaptotagmin (SYT11) in the tumor tissues of patients with GC was confirmed by performing Immunohistochemistry, and the correlation between the expression level and the patient's survival rate was analyzed. Phospho-kinase array was performed to detect Jun N-terminal kinase (JNK) phosphorylation. SYT11, JNK, and MKK7 complex formation was confirmed by western blot and immunoprecipitation assays. We studied the effects of SYT11 on GC proliferation and metastasis, real-time cell image analysis, adhesion assay, invasion assay, spheroid formation, mouse xenograft assay, and liver metastasis. RESULTS: SYT11 is highly expressed in the stem-like molecular subtype of GC in transcriptome analysis of 527 patients with GC. Moreover, SYT11 is a potential prognostic biomarker for histologically classified diffuse-type GC. SYT11 functions as a scaffold protein, binding both MKK7 and JNK1 signaling molecules that play a role in JNK1 phosphorylation. In turn, JNK activation leads to a signaling cascade resulting in cJun activation and expression of downstream genes angiopoietin-like 2 (ANGPTL2), thrombospondin 4 (THBS4), Vimentin, and junctional adhesion molecule 3 (JAM3), which play a role in epithelial-mesenchymal transition (EMT). SNU484 cells infected with SYT11 shRNA (shSYT11) exhibited reduced spheroid formation, mouse tumor formation, and liver metastasis, suggesting a pro-oncogenic role of SYT11. Furthermore, SYT11-antisense oligonucleotide (ASO) displayed antitumor activity in our mouse xenograft model and was conferred an anti-proliferative effect in SNU484 and MKN1 cells. CONCLUSION: SYT11 could be a potential therapeutic target as well as a prognostic biomarker in patients with diffuse-type GC, and SYT11-ASO could be used in therapeutic agent development for stem-like molecular subtype diffuse GC.

Glycogen Storage Disease Phenotypes Accompanying the Perturbation of the Methionine Cycle in NDRG3-Deficient Mouse Livers.

N-Myc downstream regulated gene 3 (NDRG3) is a unique pro-tumorigenic member among NDRG family genes, mediating growth signals. Here, we investigated the pathophysiological roles of NDRG3 in relation to cell metabolism by disrupting its functions in liver. Mice with liver-specific KO of NDRG3 (Ndrg3 LKO) exhibited glycogen storage disease (GSD) phenotypes including excessive hepatic glycogen accumulation, hypoglycemia, elevated liver triglyceride content, and several signs of liver injury. They suffered from impaired hepatic glucose homeostasis, due to the suppression of fasting-associated glycogenolysis and gluconeogenesis. Consistently, the expression of glycogen phosphorylase (PYGL) and glucose-6-phosphate transporter (G6PT) was significantly down-regulated in an Ndrg3 LKO-dependent manner. Transcriptomic and metabolomic analyses revealed that NDRG3 depletion significantly perturbed the methionine cycle, redirecting its flux towards branch pathways to upregulate several metabolites known to have hepatoprotective functions. Mechanistically, Ndrg3 LKO-dependent downregulation of glycine N-methyltransferase in the methionine cycle and the resultant elevation of the S-adenosylmethionine level appears to play a critical role in the restructuring of the methionine metabolism, eventually leading to the manifestation of GSD phenotypes in Ndrg3 LKO mice. Our results indicate that NDRG3 is required for the homeostasis of liver cell metabolism upstream of the glucose-glycogen flux and methionine cycle and suggest therapeutic values for regulating NDRG3 in disorders with malfunctions in these pathways.

LPIN1 Induces Gefitinib Resistance in EGFR Inhibitor-Resistant Non-Small Cell Lung Cancer Cells.

Drug resistance limits the efficacy of targeted therapies, including tyrosine kinase inhibitors (TKIs); however, a substantial portion of the drug resistance mechanisms remains unexplained. In this study, we identified LPIN1 as a key factor that regulates gefitinib resistance in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) cells. Unlike TKI-sensitive HCC827 cells, gefitinib treatment induced LPIN1 expression and increased diacylglycerol concentration in TKI-resistant H1650 cells, followed by the activation of protein kinase C delta and nuclear factor kappa B (NF-κB) in an LPIN1-dependent manner, resulting in cancer cell survival. Additionally, LPIN1 increased the production of lipid droplets, which play an important role in TKI drug resistance. All results were recapitulated in a patient-derived EGFR-mutant NSCLC cell line. In in vivo tumorigenesis assay, we identified that both shRNA-mediated depletion and pharmaceutical inhibition of LPIN1 clearly reduced tumor growth and confirmed that gefitinib treatment induced LPIN1 expression and LPIN1-dependent NF-κB activation (an increase in p-IκBα level) in tumor tissues. These results suggest an effective strategy of co-treating TKIs and LPIN1 inhibitors to prevent TKI resistance in NSCLC patients.

PKM2 Regulates HSP90-Mediated Stability of the IGF-1R Precursor Protein and Promotes Cancer Cell Survival during Hypoxia.

Insulin-like growth factor-1 receptor (IGF-1R), an important factor in promoting cancer cell growth and survival, is commonly upregulated in cancer cells. However, amplification of the IGF1R gene is extremely rare in tumors. Here, we have provided insights into the mechanisms underlying the regulation of IGF-1R protein expression. We found that PKM2 serves as a non-metabolic protein that binds to and increases IGF-1R protein expression by promoting the interaction between IGF-1R and heat-shock protein 90 (HSP90). PKM2 depletion decreases HSP90 binding to IGF-1R precursor, thereby reducing IGF-1R precursor stability and the basal level of mature IGF-1R. Consequently, PKM2 knockdown inhibits the activation of AKT, the key downstream effector of IGF-1R signaling, and increases apoptotic cancer cell death during hypoxia. Notably, we clinically verified the PKM2-regulated expression of IGF-1R through immunohistochemical staining in a tissue microarray of 112 lung cancer patients, demonstrating a significant positive correlation (r = 0.5208, p < 0.0001) between PKM2 and IGF-1R expression. Together, the results of a previous report demonstrated that AKT mediates PKM2 phosphorylation at serine-202; these results suggest that IGF-1R signaling and PKM2 mutually regulate each other to facilitate cell growth and survival, particularly under hypoxic conditions, in solid tumors with dysregulated IGF-1R expression.

LAD1 expression is associated with the metastatic potential of colorectal cancer cells.

BACKGROUND: Anchoring filament protein ladinin-1 (LAD1) was related to the aggressive progression of breast, lung, laryngeal and thyroid cancers. However, the association of LAD1 with colorectal cancer remained unknown. Here, to determine the relationship of LAD1 with colorectal cancer progression, we explored the effect of LAD1 loss on the malignant features of colorectal cancer cells. METHODS: We constructed LAD1-depleted cell lines and examined the effect of LAD1 deficiency on the phenotypic and molecular features of colorectal cancer cells in vitro. The function of LAD1 in metastasis in vivo was examined by establishing a spleen-to-liver metastasis mouse model. LAD1 protein expression in colorectal cancer patient specimens was assessed by immunohistochemistry of tumor microarrays. RESULTS: We found that LAD1 was abundant in most colorectal cancer cells. In addition, high expression of LAD1 significantly correlated with poor patient outcome. LAD1 depletion inhibited the migration and invasion of two different colorectal cancer cell lines, SW620 and Caco-2, without affecting their proliferation. In addition, LAD1 loss led to defects in liver metastasis of SW620 cells in the mouse model. Immunohistochemistry of colorectal cancer tissues revealed LAD1 enrichment in metastatic tissues compared to that in primary tumor and normal tissues. CONCLUSION: These results suggest that LAD1 expression is associated with the metastatic progression of colorectal cancer by promoting the migration and invasion of cancer cells.

A Synthetic CPP33-Conjugated HOXA9 Active Domain Peptide Inhibits Invasion Ability of Non-Small Lung Cancer Cells.

Homeobox A9 (HOXA9) expression is associated with the aggressive growth of cancer cells and poor prognosis in lung cancer. Previously, we showed that HOXA9 can serve as a potential therapeutic target for the treatment of metastatic non-small cell lung cancer (NSCLC). In the present study, we have carried out additional studies toward the development of a peptide-based therapeutic agent. Vectors expressing partial DNA fragments of HOXA9 were used to identify a unique domain involved in the inhibition of NSCLC cell invasion. Next, we performed in vitro invasion assays and examined the expression of EMT-related genes in transfected NSCLC cells. The C-terminal fragment (HOXA9-C) of HOXA9 inhibited cell invasion and led to upregulation of CDH1 and downregulation of SNAI2 in A549 and NCI-H1299 cells. Reduced SNAI2 expression was consistent with the decreased binding of transcription factor NF-kB to the SNAI2 promoter region in HOXA9-C overexpressing cells. Based on the above results, we synthesized a cell-permeable peptide, CPP33-HADP (HOXA9 active domain peptide), for lung-specific delivery and tested its therapeutic efficiency. CPP33-HADP effectively reduced the invasion ability of NSCLC cells in both in vitro and in vivo mouse models. Our results suggest that CPP33-HADP has significant potential for therapeutic applications in metastatic NSCLC.

Structural and Biophysical Analyses of Human N-Myc Downstream-Regulated Gene 3 (NDRG3) Protein.

The N-Myc downstream-regulated gene (NDRG) family belongs to the α/ß-hydrolase fold and is known to exert various physiologic functions in cell proliferation, differentiation, and hypoxia-induced cancer metabolism. In particular, NDRG3 is closely related to proliferation and migration of prostate cancer cells, and recent studies reported its implication in lactate-triggered hypoxia responses or tumorigenesis. However, the underlying mechanism for the functions of NDRG3 remains unclear. Here, we report the crystal structure of human NDRG3 at 2.2 Å resolution, with six molecules in an asymmetric unit. While NDRG3 adopts the α/ß-hydrolase fold, complete substitution of the canonical catalytic triad residues to non-reactive residues and steric hindrance around the pseudo-active site seem to disable the α/ß-hydrolase activity. While NDRG3 shares a high similarity to NDRG2 in terms of amino acid sequence and structure, NDRG3 exhibited remarkable structural differences in a flexible loop corresponding to helix α6 of NDRG2 that is responsible for tumor suppression. Thus, this flexible loop region seems to play a distinct role in oncogenic progression induced by NDRG3. Collectively, our studies could provide structural and biophysical insights into the molecular characteristics of NDRG3.

Lactate Activates the E2F Pathway to Promote Cell Motility by Up-Regulating Microtubule Modulating Genes.

Excess lactate production due to enhanced aerobic glycolysis is characteristic of malignant cancers, which is also intimately associated with poor cancer prognoses. Although tumor-associated lactate contributes to all major steps in carcinogenesis, its action mechanism remains obscure. To understand the molecular mechanism of the lactate-induced tumor metastatic process, we identified an array of lactate-responsive genes via transcriptome analysis of a metformin-induced hyper-glycolytic liver cancer model. Gene set enrichment analysis suggested E2F-RB pathway as the dominant regulator of the lactate-induced gene expression. We experimentally verified that lactate indeed activates E2F-mediated transcription by promoting E2F1 protein accumulation through a posttranscriptional mechanism. Literature-based analysis of target pathways potentially modulated by 136 top-ranked genes indicated that genes functioning in cell-cell or cell-matrix communications dominate the lactate-induced gene expression. Especially, those regulating microtubule functions, including a group of kinesin family members, were significantly up-regulated in lactate- and E2F1-dependent manners. Depletion of E2F1 or kinesins (KIF2C, KIF18B, KIF20A) led to deformation of microtubule structures, impairing cell motility as much as the deficit in lactate production. These results indicate that E2F pathway activation by tumor-associated lactate and subsequent transcriptional activation of microtubule functions play crucial roles in tumor metastasis, providing mechanistic clues to cell motility-directed anti-cancer strategies.

Recombinant cell-permeable HOXA9 protein inhibits NSCLC cell migration and invasion.

PURPOSE: Previously, it has been reported that homeobox A9 (HOXA9) protein expression is downregulated in lung cancer cells, and that its expression is inversely correlated with the metastatic potential of lung cancer cells both in vitro and in vivo. As such, HOXA9 shows therapeutic potential. The development of therapeutic strategies based on this protein is, however, limited due to its poor membrane permeability. To overcome this problem, we developed a system to deliver HOXA9 protein into non-small cell lung cancer (NSCLC) cells. METHODS: First, we constructed a delivery vector expressing polyarginine, a cell-penetrating peptide, as well as HOXA9. The resulting recombinant R10-HOXA9 protein was effectively introduced into A549 and NCI-H1299 NSCLC cells. Next, we examined the roles and molecular mechanisms of recombinant R10-HOXA9 in processes involved in tumor progression. To investigate the therapeutic efficacy of the delivery system, we performed cell motility assays using both in vitro and in vivo experimental models. RESULTS: We found that recombinant R10-HOXA9 protein reduced the invasion and migration rate, but not the proliferation rate, of the NSCLC cells tested, both in vitro and in vivo. Treatment of NSCLC cells with recombinant R10-HOXA9 protein led to a significant increase in E-cadherin expression. Conversely, we found that the expression of snail family zinc finger 2 (SLUG), a transcriptional repressor of E-cadherin, was markedly decreased. In an experimental metastatic mouse model, recombinant R10-HOXA9 protein was found to effectively reduce the rate of lung cancer cell motility. CONCLUSIONS: Our data suggest that the developed cell-permeable R10-HOXA9 system may serve as a useful tool to prevent NSCLC cell migration and invasion.

Metabolic Signaling to Epigenetic Alterations in Cancer.

Cancer cells reprogram cellular metabolism to support the malignant features of tumors, such as rapid growth and proliferation. The cancer promoting effects of metabolic reprogramming are found in many aspects: generating additional energy, providing more anabolic molecules for biosynthesis, and rebalancing cellular redox states in cancer cells. Metabolic pathways are considered the pipelines to supply metabolic cofactors of epigenetic modifiers. In this regard, cancer metabolism, whereby cellular metabolite levels are greatly altered compared to normal levels, is closely associated with cancer epigenetics, which is implicated in many stages of tumorigenesis. In this review, we provide an overview of cancer metabolism and its involvement in epigenetic modifications and suggest that the metabolic adaptation leading to epigenetic changes in cancer cells is an important non-genetic factor for tumor progression, which cooperates with genetic causes. Understanding the interaction of metabolic reprogramming with epigenetics in cancers may help to develop novel or highly improved therapeutic strategies that target cancer metabolism.

Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1α protein stability.

Oxygen deprivation induces a range of cellular adaptive responses that enable to drive cancer progression. Here, we report that lysine-specific demethylase 1 (LSD1) upregulates hypoxia responses by demethylating RACK1 protein, a component of hypoxia-inducible factor (HIF) ubiquitination machinery, and consequently suppressing the oxygen-independent degradation of HIF-1α. This ability of LSD1 is attenuated during prolonged hypoxia, with a decrease in the cellular level of flavin adenine dinucleotide (FAD), a metabolic cofactor of LSD1, causing HIF-1α downregulation in later stages of hypoxia. Exogenously provided FAD restores HIF-1α stability, indicating a rate-limiting role for FAD in LSD1-mediated HIF-1α regulation. Transcriptomic analyses of patient tissues show that the HIF-1 signature is highly correlated with the expression of LSD1 target genes as well as the enzymes of FAD biosynthetic pathway in triple-negative breast cancers, reflecting the significance of FAD-dependent LSD1 activity in cancer progression. Together, our findings provide a new insight into HIF-mediated hypoxia response regulation by coupling the FAD dependence of LSD1 activity to the regulation of HIF-1α stability.

Genome-scale functional analysis of the human genes modulating p53 activity by regulating MDM2 expression in a p53-independent manner.

MDM2, a critical negative regulator of p53, is often overexpressed in leukemia, but few p53 mutations are found, suggesting that p53-independent MDM2 expression occurs due to alterations in MDM2 upstream regulators. In this study, a high MDM2 transcription level was observed (41.17%) regardless of p53 expression in patient with acute myeloid leukemia (AML). Therefore, we performed genome-scale functional screening of the human genes modulating MDM2 expression in a p53-independent manner. We searched co-expression profiles of genes showing a positive or negative pattern with MDM2 expression in a DNA microarray database, selected1089 links, and composed a screening library of 368 genes. Using MDM2 P1 and P2 promoter-reporter systems, we screened clones regulating MDM2 transcriptions in a p53-independent manner by overexpression. Nine clones from the screening library showed enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells. Among them, six clones, including NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, and YWHAB mediated MAPK signaling for expressing MDM2. These results indicate that p53-independent upregulation of MDM2 by increasing selected clones may lead to oncogenesis in AML and that MDM2-modulating genes are novel potential targets for AML treatment.

AKT-induced PKM2 phosphorylation signals for IGF-1-stimulated cancer cell growth.

Pyruvate kinase muscle type 2 (PKM2) exhibits post-translational modifications in response to various signals from the tumor microenvironment. Insulin-like growth factor 1 (IGF-1) is a crucial signal in the tumor microenvironment that promotes cell growth and survival in many human cancers. Herein, we report that AKT directly interacts with PKM2 and phosphorylates it at Ser-202, which is essential for the nuclear translocation of PKM2 protein under stimulation of IGF-1. In the nucleus, PKM2 binds to STAT5A and induces IGF-1-stimulated cyclin D1 expression, suggesting that PKM2 acts as an important factor inducing STAT5A activation under IGF-1 signaling. Concordantly, overexpression of STAT5A in cells deficient in PKM2 expression failed to restore IGF-induced growth, whereas reconstitution of PKM2 in PKM2 knockdown cells restored the IGF-induced growth capacity. Our findings suggest a novel role of PKM2 in promoting the growth of cancers with dysregulated IGF/phosphoinositide 3-kinase/AKT signaling.

Dehydropeptidase 1 promotes metastasis through regulation of E-cadherin expression in colon cancer.

Dehydropeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is expressed aberrantly in several cancers. The role of DPEP1 in cancer remain controversial. In this study, we demonstrate that DPEP1 functions as a positive regulator for colon cancer cell metastasis. The expression of DPEP1 mRNA and proteins were upregulated in colon cancer tissues compared to normal mucosa. Gain-of-function and loss-of-function approaches were used to examine the malignant phenotype of DPEP1-expressing or DPEP1-depleted cells. DPEP1 expression caused a significant increase in colon cancer cell adhesion and invasion in vitro, and metastasis in vivo. In contrast, DPEP1 depletion induced opposite effects. Furthermore, cilastatin, a DPEP1 inhibitor, suppressed the invasion and metastasis of DPEP1-expressing cells. DPEP1 inhibited the leukotriene D4 signaling pathway and increased the expression of E-cadherin. We also show that DPEP1 mediates TGF-ß-induced EMT. TGF-ß transcriptionally repressed DPEP1 expression. TGF-ß treatment decreased E-cadherin expression and promoted cell invasion in DPEP1-expressing colon cancer cell lines, whereas it did not affect these parameters in DPEP1-depleted cell lines. These results suggest that DPEP1 promotes cancer metastasis by regulating E-cadherin plasticity and that it might be a potential therapeutic target for preventing the progression of colon cancer.

High-resolution transcriptome analysis reveals neuropathic pain gene-expression signatures in spinal microglia after nerve injury.

Microglial cells, the resident immune cells of the spinal cord, become activated in response to peripheral nerve injury. Microglia activation contributes to the development of neuropathic pain. Here we employed microarray analysis of individually collected pools of 10 spinal microglia cells to identify changes of levels and cell-to-cell expression variance of microglial genes during their activation after peripheral nerve injury. The analysis of microglia on postoperative day 1 (POD1) identified miR-29c as a critical factor for microglial activation and the development of neuropathic pain. Early POD1 microglia exhibited a very distinct expression profile compared to late POD7 microglia, possibly leading to the transition from initiation to maintenance of neuropathic pain. We found sample variance patterns that were consistent with the hypothesis that microglia were highly heterogeneous at the level of individual cells, and variation analysis identified 56 microglial genes potentially linked to the maintenance of neuropathic pain which included Gria1. This study provides insights into spinal microglial biology and reveals novel microglial targets for the treatment of neuropathic pain.

Homeobox A9 directly targeted by miR-196b regulates aggressiveness through nuclear Factor-kappa B activity in non-small cell lung cancer cells.

MicroRNAs (miRNAs) are recognized as crucial posttranscriptional regulators of gene expression, and play critical roles as oncogenes or tumor suppressors in various cancers. Here, we show that miR-196b is upregulated in mesenchymal-like-state non-small cell lung cancer (NSCLC) cells and lung cancer tissues. Moreover, miR-196b upregulation stimulates cell invasion and a change in cell morphology to a spindle shape via loss of cell-to-cell contacts. We identified homeobox A9 (HOXA9) as a target gene of miR-196b by using public databases such as TargetScan, miRDB, and microRNA.org. HOXA9 expression is inversely correlated with miR-196b levels in clinical NSCLC samples as compared to that in corresponding control samples, and with the migration and invasion of NSCLC cells. Ectopic expression of HOXA9 resulted in a suppression of miR-196b-induced cell invasion, and HOXA9 reexpression increased E-cadherin expression. Furthermore, HOXA9 potently attenuated the expression of snail family zinc finger 2 (SNAI2/SLUG) and matrix metallopeptidase 9 (MMP9) by controlling the binding of nuclear factor-kappa B to the promoter of SLUG and MMP9 genes, respectively. Therefore, we suggest that HOXA9 plays a central role in controlling the aggressive behavior of lung cancer cells and that miR-196b can serve as a potential target for developing anticancer agents. © 2015 Wiley Periodicals, Inc.

Herbacetin Is a Novel Allosteric Inhibitor of Ornithine Decarboxylase with Antitumor Activity.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials.

Pyruvate kinase isoenzyme M2 is a therapeutic target of gemcitabine-resistant pancreatic cancer cells.

Despite its wide use as a first-line therapeutic agent, gemcitabine has shown limited efficacy in advanced pancreatic cancer due to chemoresistance by as yet unidentified mechanisms. Our goal here was to identify molecular features involved in gemcitabine chemoresistance. Pyruvate kinase M2 (PKM2), a key enzyme of aerobic glycolysis, has recently emerged as an important therapeutic target for cancer treatment. It is involved in the metabolic reprogramming of cancer cells and has previously unexpected non-metabolic functions that are heavily involved in tumor growth and survival. Herein, we report that the chemoresistance of pancreatic cancer to gemcitabine was dependent on PKM2 expression and its non-metabolic function. Knocking-down of PKM2 significantly enhanced gemcitabine-induced cell apoptosis through the activation of caspase 3/7 and PARP cleavage, and this inhibitory activity was associated with p38-mediated activation of p53 phosphorylation at serine 46. Our findings support the potential of PKM2 as a novel target for gemcitabine chemoresistance and suggest the feasibility of combining gemcitabine and PKM2 inhibition for the improved chemotherapy of pancreatic cancer.

NDRG3-mediated lactate signaling in hypoxia.

Hypoxia is associated with many pathological conditions as well as the normal physiology of metazoans. We identified a lactate-dependent signaling pathway in hypoxia, mediated by the oxygen- and lactate-regulated protein NDRG family member 3 (NDRG3). Oxygen negatively regulates NDRG3 expression at the protein level via the PHD2/VHL system, whereas lactate, produced in excess under prolonged hypoxia, blocks its proteasomal degradation by binding to NDRG3. We also found that the stabilized NDRG3 protein promotes angiogenesis and cell growth under hypoxia by activating the Raf-ERK pathway. Inhibiting cellular lactate production abolishes NDRG3-mediated hypoxia responses. The NDRG3-Raf-ERK axis therefore provides the genetic basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases in addition to advancing our understanding of the normal physiology of hypoxia responses.